1. Field of the Invention
The present invention relates generally to polypeptide compositions having active functionalities covalently attached proximate a binding site. More particularly, the invention relates to polypeptides wherein the active functionality is a reporter molecule or a chemotherapeutic agent, and to methods for the preparation of such polypeptides.
Numerous biological proteins are capable of specific attachment to other biological molecules as a result of intermolecular binding forces, including electrostatic bonds, hydrogen bonds, and van der Waals bonds. Such bonding occurs at a site on the protein generally referred to as a binding site, and binding occurs only with target ligands having a specific molecular geometry which is complementary to the binding site. Examples of such interactions include antibody-antigen binding, enzyme-substrate binding, hormone-receptor binding, lymphokine-lymphokine receptor binding, and the like. The availability of such specific binding interactions is relied on in a variety of scientific, medical, and commercial processes, including immunoassays, therapeutic treatments, immunohistochemical methods, protein separations, reaction catalysis through transition state stabilization, and the like.
In many instances, it would be desirable to equip a specific binding protein or polypeptide with a constituent or functionality proximate the binding site, where the constituent will be able to provide a desired interaction with the target ligand. For example, it will often be desirable to provide constituents which are detectable or which have therapeutic or catalytic activity proximate a polypeptide binding site specific for a particular target ligand. By providing chemotherapeutic agents proximate the binding site (and absent from other locations) on specific binding proteins capable of binding diseased cells, chemotherapeutic activity may be enhanced while reducing toxic and other side effects which might otherwise be present. By providing a reporter molecule proximate a protein binding site (and absent from other locations) specific for a particular substance, assays may be provided where the presence of substrate provides for direct modulation of the observed signal from the reporter molecule.
Heretofore, methods for covalently binding active functionalities onto specific binding proteins, such as antibodies, have generally not been localized near the binding site. That is, binding has been achieved through amino acid side chains and carbohydrates which may be found at various locations on the protein surface, not limited to locations proximate the binding site. While enzymes have been modified to include functionalities, such as cofactors, proximate active sites, the localized binding of reporter molecules and chemotherapeutic agents proximate a protein binding site has not generally been taught. Moreover, methods for localized binding of active functionalities to proteins which have not been structurally characterized are generally unknown, regardless of the type of protein and type of active functionality.
In view of the above, it would be desirable to provide polypeptides having binding sites wherein chemotherapeutic agents or reporter molecules are bound proximate the binding site and substantially absent from the remainder of the polypeptide. It would be further desirable to provide methods for covalently binding active functionalities, including reporter molecules, chemotherapeutic agents, catalytic groups, reactive groups, and the like, proximate the binding site on polypeptides, even when the structure of the polypeptide is unknown.
2. Description of the Relevant Art
Immunotoxins comprising toxins and drugs covalently bound to antibodies specific for cellular receptors and other targets are known. See, e.g., U.S. Pat. Nos. 4,340,535; 4,368,149; 4,489,710; and 4,671,958. None of these patents, however, disclose site-specific binding of the toxins and drugs proximate the antibody binding site. European patent application 187 658 discloses antibodies covalently attached to activators. The antibodies bind to diseased sites within a host and the activators interact with an inactive substance within the host to produce an active substance. Binding of the activators is not limited to the antibody binding site. U.S. Pat. No. 3,817,752, describes homogeneous enzyme immunoassays employing detectable enzymes having hapten bound thereto. By exposing the enzymes to antibodies specific for the hapten, modulation of the enzyme activity can be achieved. The use of spin labels bound to enzymes to study enzyme activity is disclosed in Jones et al. (1973) Eur. J. Biochem. 34:28-40. Polyclonal antibodies have been generated with cofactor binding sites (Raso and Stollar (1975) Biochemistry 14:584-591). The generation of monoclonal antibodies having a cofactor binding site is described in Shokat et al. (1988) Angew. Chem. 100:1227-1229. The generation of affinity labels for antibody combining sites is described by Kohen et al. (1980) FEBS Lett. 11:427-431; Coetzl et al. (1970) Biochemistry 1267-1278; and Givol et al. (1971) Biochemistry 10:3461-3466. Site-directed mutagenesis has been used in conjunction with high resolution x-ray crystallography to analyze the structure of enzyme binding sites and the function of such sites in catalysis. Wilkinson et al. (1984) Nature 307:187-188; Craik et al. (1985) Science 228:291-297; Schultz et al. (1985) Biochemistry 24:6840-6848; Dalbadie-McFarland (1982) Proc. Natl. Acad. Sci. USA 79:6409-6413; and Sigal et al. (1984) J. Biol. Chem. 259:5327-5332. A cysteine in the active region of the enzyme papain has been modified with a flavin cofactor. Kaiser et al. (1984) Science 226:505-510. A thiol has been introduced into the enzymes staphylococcal nuclease and RNase S and subsequently derivatized with an oligonucleotide. Corey et al. (1987) Science 38:1401-1403. The active site serine of subtilisin has been chemically converted to a cysteine. Bender et al. (1966) J. Am. Chem. Soc. 88:3153-3154 and Koshland et al. (1966) Proc. Natl. Acad. Sci. USA 56:1606-1611.